Legionella pneumophila is a bacterial pathogen that causes a severe form of pneumonia in human beings. L. pneumophila is taken up by macrophages and replicates inside the cell within a membrane-bound compartment called a phagosome. L. pneumophila subverts the normal phagocytic pathway and converts the phagosome into a site suitable for bacterial replication. The broad objective of this proposal is to identify the bacterial and host-cell proteins associated with the phagosomal membrane and to determine their importance in the intracellular growth and targeting of L. pneumophila genes. To this end the following experiments will be performed: 1) Antibodies will be raised against bacterial proteins on the surface of the phagosome. The antibodies will be used to isolate corresponding L. pneumophila genes by probing an expression library of L. pneumophila DNA. 2) L. pneumophila containing phagosomes will be isolated. The purified phagosomes will be probed with antibodies to proteins involved in vesicle trafficking, with antibodies raised in Specific Aim 1, and with antibodies raised against previously identified bacterial factors that are important for intracellular growth. 3) A non-prejudiced search for host proteins associated with phagosomes will be performed by purifying phagosomes form radioactively labeled host cells and analyzing the associated proteins by 2-D gel electrophoresis. The proteins will be identified by peptide sequencing. 4) Changes in bacterial and host proteins associated with the phagosome will be examined during the course of the infection using the technology developed in the previous Aims.